The present invention generally relates to the preparation of compounds useful in the semi-synthesis of taxanes. More particularly, the present invention is directed to the chiral resolution of mixtures of optical isomers to provide a target chiral compound that can be used as a C-13 side chain precursor to produce paclitaxel and other taxanes. The present invention specifically provides a chromatographic process for separating a target chiral compound from its enantiomer in a racemic mixture thereof.
Various taxane compounds are known to exhibit anti-tumor activity. As a result of this activity, taxanes have received increasing attention in the scientific and medical community. Primary among these is a compound known as xe2x80x9cpaclitaxelxe2x80x9d which is also referred to in the literature as xe2x80x9ctaxolxe2x80x9d. Paclitaxel has been approved for the chemotherapeutic treatment of several different varieties of tumors, and the clinical trials indicate that paclitaxel promises a broad range of potent anti-leukemic and tumor-inhibiting activity.
Paclitaxel is a naturally occurring taxane diterpenoid which is found in several species of the yew (genus Taxus, family Taxaceae). Unfortunately, the concentration of this compound in the yew is very low, and the species of evergreen are also slow growing. Even though the bark of the yew trees typically exhibits the highest concentration of paclitaxel, the production of one kilogram of paclitaxel requires approximately 16,000 pounds of bark. Thus, the long-term prospects for the availability of paclitaxel through isolation are discouraging.
While the presence of paclitaxel in the yew tree is in extremely low concentrations, there are a variety of other taxane compounds, such as baccatin III, cephalomanine, 10-deacetylbaccatin III, etc., which are also able to be extracted from the yew bark and leaves. Some of these other taxane compounds are more readily extracted in higher yields. Indeed, a relatively high concentration of 10-deacetylbaccatin III can be extracted from the leaves of the yew as a renewable resource.
Accordingly, attention has turned to the semi-synthesis of paclitaxel, which has the formula: 
as well as other related taxanes, such as docetaxel, which has the formula: 
from precursor compounds. In order to successfully synthesize paclitaxel and other taxanes, convenient access to a chiral, non-racemic side chain acid is desired, as well as an abundant natural source of a usable baccatin III backbone. Various approaches have been developed for esterifying such a side chain acid at the 13-hydroxyl of baccatin III or 10-deacetyl baccatin III, or derivatives thereof, which respectively have the formulas: 
The coupled ester product may then be converted to paclitaxel, docetaxel or other taxanes. For example, U.S. Pat. No. 4,924,011 to Denis et al. describes a process for preparing paclitaxel using a (2R,3S) side chain acid of the general formula: 
where R2 is a hydroxy-protecting group. Additionally, U.S. Pat. No. 4,924,012 to Colin et al. describes a process for preparing taxanes, such as docetaxel, that uses an acid of formula: 
where R1 is a hydroxy-protecting group. In the syntheses described by Denis et al. and Colin et al., the side chain acid is esterified with a baccatin III or 10-deacetyl baccatin III derivative, and the coupled product is thereafter deprotected, such as by replacing any protecting groups, including R2 or R1 in the above formulas, respectively, with hydrogen. It should be noted that the C6H5CONHxe2x80x94 group of Denis et al. and the (CH3)3COCONHxe2x80x94 group of Colin et al. are the final desired groups for the resulting paclitaxel and docetaxel products, respectively, such that no further chemical transformation at the nitrogen position is performed in the chemical syntheses disclosed therein.
U.S. Pat. No. 5,770,745 to Swindell et al. describes another early synthetic route in the semi-synthesis of paclitaxel, wherein the use of protecting groups to protect various positions of the taxane backbone and the side chain acid was investigated as a means of improving the chemical process to form paclitaxel, and of improving the esterification step in particular. Specifically, a side chain acid of the general formula: 
is described, wherein R1 can be an alkyl, olefinic or aromatic group (such as PhCH2), R3 can be hydrogen or Ph, and P1 can be a hydroxyl protecting group.
More recently, attention has been focused on the use of a 3-N-CBZ-2-O-protected (2R,3S)-3-phenylisoserine acid of the formula: 
wherein P1 is a hydrogenatable protecting group, such as benzyloxymethyl (BOM) or benzyl. Synthetic routes to produce taxanes such as paclitaxel or docetaxel using such a side chain are described, for example, in U.S. Pat. Nos. 5,675,025; 5,684,175; 5,688,977; 5,750,737; 5,939,566; 5,948,919; 5,973,170; 6,048,990; 6,066,749; 6,072,060; 6,107,497; 6,133,462; 6,136,999; and 6,143,902, and the teachings thereof are incorporated herein by reference.
As taught, for example, in U.S. Pat. No. 5,684,175 to Sisti et al., the 3-N-CBZ-2-O-protected (2R,3S)-3-phenylisoserine may be produced from a (2R,3S)-3-phenylisoserine ethyl ester starting compound of the formula: 
which may be protected at the 3xe2x80x2-N and 2xe2x80x2-O positions and saponified to the corresponding acid for use in the esterification step.
The formation of a 3-phenylisoserine alkyl ester is known in the art and is described, for example, in U.S. Pat. No. 4,924,012 to Colin et al. In particular, Colin et al. describes an epoxide of the formula: 
where R4 denotes alkyl containing 1 to 4 carbon atoms, and preferably ethyl, which may be obtained under the conditions described by F. W. Bachelor and R. K. Bansal, J. Org. Chem., 34, 3600-04 (1969). This epoxide is converted to an azide of general formula: 
according to known methods for opening an epoxide by means of sodium azide in ethanol in the heated state. The azide is thereafter reduced to a 3-phenylisoserine alkyl ester of the formula: 
However, because this compound has two chiral centers (C-2 and C-3, respectively), processes to produce this compound may produce both the cis (2R,3S) and (2S,3R) enantiomers respectively, such as of the formulas: 
as well as their trans (2R,3R) and (2S,3S) diastereomers respectively of the formulas: 
It should be appreciated that the above optical isomers are shown using an ethyl group for R4 in the formulas above, although other alkyl esters as described in Colin et al. may be formed. Additionally, various other derivatives or analogs of these optical isomers may be formed in the processes to produce the paclitaxel side chain, in view of the teachings of the above-identified patents, as would be understood by the ordinarily skilled person.
While baccatin III and derivatives thereof may be used as a resolving agent to selectively recover products having the desired (2R,3S) configuration, such an approach is quite costly and results in a substantial yield reduction of the desired product. Accordingly, efficient and economical methods are needed for recovering a desired (2R,3S) isomer or a derivative thereof at one or more points in the chemical processes for synthesizing taxanes.
One procedure to prepare the chiral (2R,3S) C-13 side chain of paclitaxel is described by Srivastava and McChesney, xe2x80x9cA Practical and Inexpensive Synthesis of the Taxol C-13 Side Chain; N-Benzoyl-(2R,3S)-3-Phenylisoserinexe2x80x9d, Natural Products Letters, Vol. 6, p. 147 (1995). This procedure involves the classical resolution of cis-3-phenylglycidic acid using D-(+)-ephedrine as the resolving agent. In particular, an optically pure cis-(2R,3R)-3-phenylglycidic acid-(+)-ephedrine salt of the formula: 
is recovered from a mixture of diastereomeric salts by fractional crystallization with acetone. The optically pure intermediate is then converted to a 3-phenylisoserine derivative. Additional classical resolutions of side chain derivatives useful in paclitaxel synthesis have been described in U.S. Pat. No. 6,025,516 to Ramaswamy et al. and in U.S. Pat. No. 5,817,867 to Li.
Several other procedures are reported in the literature to prepare chiral, cis-3-phenylglycidate derived intermediates useful in the partial synthesis of paclitaxel. Some examples include methodologies utilizing Sharpless asymmetric dihydroxylation (Denis et al., J. Org. Chem., 55, p.1957, 1990; Koskinen et al., J. Chem. Soc., Chem. Commun., p.21, 1994; and Bonini et al., J. Chem. Soc., Chem. Commun., p.2767, 1994) and some utilizing Jacobsen""s asymmetric epoxidation (Deng et al., J. Org. Chem., 57, p.4323, 1992). Other references have reported chemoenzymatic resolution methodologies (Chen et al., J. Org. Chem., 58, p.1287; U.S. Pat. No. 6,020,174 to Chen et al.; Honig et al., Tetrahedron Letters, 46, p.3841, 1990; U.S. Pat. No. 5,773,629 to Yang et al.; and U.S. Pat. Nos. 5,811,292, 5,567,614 and 5,686,298 to Patel et al).
However, there remains a need to provide new and improved methods for the efficient and economical preparation of chiral compounds useful in taxane semi-synthesis. In particular, there remains a need for simple methods for producing chiral compounds useful as precursors for the C-13 side-chain esterification with a baccatin III backbone. The present invention is directed to meeting these needs.
It is an object of the present invention to provide a new and useful method for preparing chiral compounds useful in taxane semi-synthesis.
It is another object to provide methods for preparing optically pure derivatives of 3-phenylisoserine.
It is yet another object to provide for simple and efficient chiral chromatographic separation of optical isomers useful in the synthesis of taxanes such as paclitaxel and docetaxel.
A still further object is to provide chromatography media useful in chromatographically separating optical isomers of 3-phenylisoserine derivatives.
Yet another object is to synthesize an N-CBZ-(2R,3S)-3-phenylisoserine side chain derivative in optically pure form that is suitable for coupling with a suitable baccatin III derivative to provide paclitaxel after various synthetic transformations.
According to the present invention, a method is provided for processing a solution having a plurality of optical isomers, such as a racemic solution, thereby to obtain a (2R,3S) target isomer having a formula: 
wherein P1 is selected from H and a hydroxyl protecting group, R1 is selected from H, an alkyl group, an olefinic group and an aromatic group, and R2 is selected from H and R3CO, where R3 is selected from an alkyl group, an olefinic group, an aromatic group, an O-alkyl group, an O-olefinic group and an O-aromatic group. The method comprises the steps of passing the solution through a chromatographic stationary phase that has a greater affinity for either the target isomer or an optical isomer thereof, such that the target isomer passes through the stationary phase at a different rate than does the optical isomer thereof, and thereafter collecting a portion of the solution containing the target isomer.
The stationary phase may be loaded into a chromatography column having a first opening and a second opening, and the step of passing the solution through the stationary phase may be accomplished by injecting the solution into the first opening. The portion of the solution containing the target isomer may be collected from the second opening. Preferably, the solution contains the target isomer and the optical isomer thereof in a first ratio to one another, and the collected portion of the solution contains the target isomer and the optical isomer thereof in a second ratio to one another that is greater than the first ratio.
The stationary phase may include tetrahydrophenanthrene xcfx80 systems, dinitrobenzamide xcfx80 systems and amido-proton hydrogen bond donors, and preferably comprises S,S Whelk-O which may be covalently bonded to silica. The solution may include ethanol, isopropanol, hexane or the like, and preferably includes 20% isopropanol/80% hexane, and may contain about 0.025 g of the optical isomers per 1 mL isopropanol.
Various optical isomers are contemplated, including ones wherein R1 is an alkyl group, R2 is R3CO where R3 is PhCH2O, and wherein P1 is hydrogen, and particularly a (2R,3S)-N-CBZ-3-phenylisoserine ethyl ester of the formula: 
The present invention particularly contemplates a method of processing a racemic mixture of (xc2x1)-N-CBZ-3-phenylisoserine ethyl ester thereby to separate the (2R,3S) isomer from the enantiomer thereof, comprising chromatographing the racemic mixture by HPLC using a stationary phase comprising S,S Whelk-O and a mobile phase including ethanol, isopropanol or hexane. The HPLC peak for the (2R,3S) isomer is preferably resolved to the baseline from the HPLC peak for the enantiomer thereof.
Further, the invention may broadly include a method of processing optical isomers of the general formula: 
wherein P1 is H or a hydroxyl protecting group, R1 is selected from H, an alkyl group, an olefinic group and an aromatic group, and R2 is selected from H and R3CO, where R3 is selected from an alkyl group, an olefinic group, an aromatic group, an O-alkyl group, an O-olefinic group and an O-aromatic group, thereby to separate the optical isomers from one another, comprising chromatographing the racemic mixture by HPLC using a stationary phase that has a different affinity for each of the optical isomers.
These and other objects of the present invention will become more readily appreciated and understood from a consideration of the following detailed description of the exemplary embodiments of the present invention when taken together with the accompanying drawings, in which: